Column: CROWNPAK CR-I (-) (Daicel Corporation) Size: 150 × 3 mm ID
Eluent A: 20 mM HClO4 in 70:30 (v/v) mixture of H2O and ACN
Eluent B: 20 mM HClO4 in 10:90 (v/v) mixture of H2O and ACN
Eluent C: 100/100/4/1 (v/v/v/v) mixture of n-hexane, ethanol, water, and trifluoroacetic acid
Flow rate: 0.25 mL/min., Temperature: 30 °C, and Detection: UV 254 nm under all conditions.
V0 was determined with formamide as the marker to be 0.809 mL.